The centromere-kinetochore complex (CKC) mediates high fidelity chromosomal segregation, owing to both its mechanical and regulatory roles in orchestrating mitosis. It safeguards against aneuploidy associated with tumorigenesis, miscarriages, and congenital disorders. The pathway of molecular interactions leading to CKC assembly on human chromosomes remains largely unknown. The long range objectives of this project are to elucidate CKC specification, assembly, and function in a model organism, S. cerevisiae, using the yeast homologs of human CENP-A and CENP-C-Cse4p and Mif2p-as focal points. The specific aims are: 1. To elucidate budding yeast CKC assembly and architecture in vivo. a) Yeast centromere protein-CEN DNA interactions, as defined by in vivo crosslinking and immunoprecipitation (ChIP), will be assessed in mutants defective for cis- or trans-acting centromere components and in synchronized cells to generate a genetic and temporal blueprint for centromere assembly. b) ChIP will also be used to test whether yeast mitotic checkpoint pathway proteins, components of a signaling pathway that responds to centromere defects, are present at CKCs. 2. To develop additional assays for structural analysis of CKCs assembled in vivo. a) UV crosslinking will be used to characterize direct centromere protein-CEN DNA interactions. b) A CEN DNA-based one-hybrid system will be developed to study centromere protein-CEN DNA interactions in vivo independently of crosslinking. 3. To define the intrinsic and extrinsic determinants of Cse4p and Mif2p CKC targeting. a) Cse4p and Mif2p interaction with other yeast CKC proteins will be tested using a two-hybrid approach. b) Deletion analysis will be used to map domains required for two-hybrid interactions and centromere targeting as determined by ChIP. 4. To characterize the function of the CENP-C-related centromere protein, Mif2p. a) mif2 mutants will be tested for centromere-specific defects using genetic, cytological and biochemical assays. b) In vitro translated or recombinant Mif2p will be tested for in vitro DNA and MT binding. And c) proteins that interact with Mif2p will be identified using two-hybrid and genetic approaches. 5. To use Cse4p as a cytological marker for yeast centromere position. Cse4p localization will be characterized using indirect immunofluorescence, GFP fluorescence, and immuno-EM in wildtype and mutant cells.